Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vecto...Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoris KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mgL-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 17 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.展开更多
To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells （PBMC）...To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells （PBMC） by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis. The expression vector pET-30a/TRAIL was then constructed by DNA recombination method with a His-tag gene at the front of the TRAIL fragment, and the recombinant protein was expressed in E. coli BL21 （DE3）. Meanwhile, the expressed target protein was purified with Ni-NTA chromatography column and identified by SDS-PAGE and Western blotting. The proliferation inhibition activity of TRAIL-His was detected by MTF assay. PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-induced cell apoptosis. It was demonstrated that the target protein expressed in E. coli BL21 showed the same relative molecular mass as that the protein expected and could be recognized by both the anti-TRAIL polyclonal antibody and anti-His monoclonal antibody. In addition, this protein could also inhibit proliferation of human lymphoma cell line Jurkat cells or induce apoptosis of this cell line. It is apparent that a recombinant soluble TRAIL protein with biological activity is obtained and this prospective study can lay solid foundation for further research on the biological activity and application in the anti-tumor therapy.展开更多
With the announcement of the sheep clone Dolly as the breakthrough in the biotechnology in news media around the turn of the twenty-first century, the rising issue of human clones in its development and the controvers...With the announcement of the sheep clone Dolly as the breakthrough in the biotechnology in news media around the turn of the twenty-first century, the rising issue of human clones in its development and the controversially bioethical issues ensued, Kazuo Ishiguro in Never Let Me Go （2005） focuses his attention, in the area of cell therapy, on how human clones, since produced, lead their model lives and face their deaths, in order that his readers may better understand the meanings of life and death, and that they may stay in a far closer relationship with their family and friends than ever. In this essay, I examine, in two worlds, the normals＇ and the clones＇, paralleling each other, the true meanings of being human and their lives through the perspective of Jacques Derrida＇s deconstruction; and I argue that Ishiguro misspeaks to his readers the true meanings of life and death especially through the clones＇ perspective and brings them to his readers＇ hearts further realistically. In Derrida＇s nature-culture structurality of the clones, it is Kathy H. who comes as center into which the other clones come as freeplay in the structurality of the real world, where it is normals who come as center into which clones come as freeplay under the structurality of power in the institutions where the clones＇ culture comes as center into which Miss Emily＇s ruling comes as freeplay by the structurality of authorship where the author comes as center into which the novel comes as freeplay.展开更多
基金the National Natural Science Foundation of China (No.19732003)
文摘Pichia pastoris expression system is similar to that of the mammal cell in modification of expressed protein, including refolding and glycosylation. A human aFGF gene was cloned into the intracellular expression vector pPIC9K. The Pichia pastoris KM71 strain was transformed with the recombined expression plasmid. Transgenic expression was observed after screening the transformants with G418. The expression and secretion of recombinant human aFGF (rhaFGF) into the culture medium were testified by ELISA assay. The yield peaked after two days of induction and was approximately 10 mgL-1 in shake-flask fermentation medium. The recombinant proteins were purified by the combination of heparin-Sepharose affinity chromatography and gel filtration chromatography. Two proteins with relative molecular masses (Mr) of 17 000 and 35 000 were purified as a single band in SDS-PAGE, whose biological activities were determined by MTT assay. It is found that the protein with Mr of 17 000 is nonglycosylated haFGF, and that with Mr of 35 000 is glycosylated haFGF; and the latter has a lower biological activity than the former.
文摘To obtain the recombinant soluble protein of the extracellular fragment of human TRAIL gene and to identify its function preliminarily, this gene fragment was amplified from peripheral blood mononuclear cells （PBMC） by RT-PCR and cloned into vector pGEM-T-Easy for sequence analysis. The expression vector pET-30a/TRAIL was then constructed by DNA recombination method with a His-tag gene at the front of the TRAIL fragment, and the recombinant protein was expressed in E. coli BL21 （DE3）. Meanwhile, the expressed target protein was purified with Ni-NTA chromatography column and identified by SDS-PAGE and Western blotting. The proliferation inhibition activity of TRAIL-His was detected by MTF assay. PI staining and Wright-Giemsa staining were used to detect the presence of the TRAIL-induced cell apoptosis. It was demonstrated that the target protein expressed in E. coli BL21 showed the same relative molecular mass as that the protein expected and could be recognized by both the anti-TRAIL polyclonal antibody and anti-His monoclonal antibody. In addition, this protein could also inhibit proliferation of human lymphoma cell line Jurkat cells or induce apoptosis of this cell line. It is apparent that a recombinant soluble TRAIL protein with biological activity is obtained and this prospective study can lay solid foundation for further research on the biological activity and application in the anti-tumor therapy.
文摘With the announcement of the sheep clone Dolly as the breakthrough in the biotechnology in news media around the turn of the twenty-first century, the rising issue of human clones in its development and the controversially bioethical issues ensued, Kazuo Ishiguro in Never Let Me Go （2005） focuses his attention, in the area of cell therapy, on how human clones, since produced, lead their model lives and face their deaths, in order that his readers may better understand the meanings of life and death, and that they may stay in a far closer relationship with their family and friends than ever. In this essay, I examine, in two worlds, the normals＇ and the clones＇, paralleling each other, the true meanings of being human and their lives through the perspective of Jacques Derrida＇s deconstruction; and I argue that Ishiguro misspeaks to his readers the true meanings of life and death especially through the clones＇ perspective and brings them to his readers＇ hearts further realistically. In Derrida＇s nature-culture structurality of the clones, it is Kathy H. who comes as center into which the other clones come as freeplay in the structurality of the real world, where it is normals who come as center into which clones come as freeplay under the structurality of power in the institutions where the clones＇ culture comes as center into which Miss Emily＇s ruling comes as freeplay by the structurality of authorship where the author comes as center into which the novel comes as freeplay.
